Inhibition of aldose reductase prevents lipopolysaccharide-induced inflammatory response in human lens epithelial cells

Agnieszka Pladzyk, Aramati B M Reddy, Umesh C S Yadav, Ravinder Tammali, Kota Ramana, Satish Srivastava

Research output: Contribution to journalArticle

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Abstract

PURPOSE. Bacterial infections are one of the major causes of human eye disease. Because the bacterial endotoxin lipopolysaccharide (LPS) is known to cause cytotoxicity through oxidative stress and an earlier study has shown that aldose reductase (AR) mediates oxidative stress signals, the purpose of this study was to investigate the anti-inflammatory effects of AR inhibition on LPS-induced activation of NF-κB- dependent signals in human lens epithelial cells (HLECs). METHODS. Growth-arrested HLECs were cultured without or with AR inhibitors or transfected with an AR small interfering (si)RNA. Subsequently, the cells were stimulated with LPS (1-10 μg/mL) for 24 hours. The cell viability was assessed by cell counts and MTT assay, and apoptosis was measured by nucleosomal degradation. Electrophoretic mobility gel shift assays were performed to determine the activation of NF-κB and AP1. The levels of nitric oxide, MMP-2, MMP-9, Cox-2, and TNF-α were measured by using specific ELISA kits. Western blot analysis was performed to determine the cleavage of poly(ADP-ribose) polymerase (PARP) and the activation of PKC and mitogen-activated protein kinase (MAPK). RESULTS. Bacterial LPS caused apoptosis of HLECs. Inhibition of AR by two structurally unrelated inhibitors, sorbinil and tolrestat, or ablation by AR siRNA prevented the LPS-induced apoptosis, activation of caspase-3 and cleavage of PARP protein. Inhibition of AR in HLECs also prevented the LPS-induced activation of redox-sensitive transcription factors such as NF-κB and AP1 and their downstream signals that lead to expression of Cox-2, MMP-2, MMP-9, and TNF-α proteins. In addition, inhibition of AR prevented LPS-induced activation of protein kinases upstream to NF-κB activation such as PKC and MAPK in HLECs. CONCLUSIONS. The results indicate that AR mediates the bacterial endotoxin signaling that could damage HLECs by regulating the signals that activate the redox-sensitive transcription factor NF-κB and cause inflammation. Thus, inhibition of AR could be a therapeutic target for Gram-negative bacterial infection-induced visual complications.

Original languageEnglish (US)
Pages (from-to)5395-5403
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume47
Issue number12
DOIs
StatePublished - Dec 2006

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Aldehyde Reductase
Lenses
Lipopolysaccharides
Epithelial Cells
Matrix Metalloproteinases
Poly(ADP-ribose) Polymerases
Apoptosis
Mitogen-Activated Protein Kinases
Endotoxins
Small Interfering RNA
Oxidation-Reduction
Oxidative Stress
Transcription Factors
Gram-Negative Bacterial Infections
Eye Diseases
Electrophoretic Mobility Shift Assay
Bacterial Infections
Caspase 3
Protein Kinases
Cell Survival

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Inhibition of aldose reductase prevents lipopolysaccharide-induced inflammatory response in human lens epithelial cells. / Pladzyk, Agnieszka; Reddy, Aramati B M; Yadav, Umesh C S; Tammali, Ravinder; Ramana, Kota; Srivastava, Satish.

In: Investigative Ophthalmology and Visual Science, Vol. 47, No. 12, 12.2006, p. 5395-5403.

Research output: Contribution to journalArticle

Pladzyk, Agnieszka ; Reddy, Aramati B M ; Yadav, Umesh C S ; Tammali, Ravinder ; Ramana, Kota ; Srivastava, Satish. / Inhibition of aldose reductase prevents lipopolysaccharide-induced inflammatory response in human lens epithelial cells. In: Investigative Ophthalmology and Visual Science. 2006 ; Vol. 47, No. 12. pp. 5395-5403.
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AU - Tammali, Ravinder

AU - Ramana, Kota

AU - Srivastava, Satish

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N2 - PURPOSE. Bacterial infections are one of the major causes of human eye disease. Because the bacterial endotoxin lipopolysaccharide (LPS) is known to cause cytotoxicity through oxidative stress and an earlier study has shown that aldose reductase (AR) mediates oxidative stress signals, the purpose of this study was to investigate the anti-inflammatory effects of AR inhibition on LPS-induced activation of NF-κB- dependent signals in human lens epithelial cells (HLECs). METHODS. Growth-arrested HLECs were cultured without or with AR inhibitors or transfected with an AR small interfering (si)RNA. Subsequently, the cells were stimulated with LPS (1-10 μg/mL) for 24 hours. The cell viability was assessed by cell counts and MTT assay, and apoptosis was measured by nucleosomal degradation. Electrophoretic mobility gel shift assays were performed to determine the activation of NF-κB and AP1. The levels of nitric oxide, MMP-2, MMP-9, Cox-2, and TNF-α were measured by using specific ELISA kits. Western blot analysis was performed to determine the cleavage of poly(ADP-ribose) polymerase (PARP) and the activation of PKC and mitogen-activated protein kinase (MAPK). RESULTS. Bacterial LPS caused apoptosis of HLECs. Inhibition of AR by two structurally unrelated inhibitors, sorbinil and tolrestat, or ablation by AR siRNA prevented the LPS-induced apoptosis, activation of caspase-3 and cleavage of PARP protein. Inhibition of AR in HLECs also prevented the LPS-induced activation of redox-sensitive transcription factors such as NF-κB and AP1 and their downstream signals that lead to expression of Cox-2, MMP-2, MMP-9, and TNF-α proteins. In addition, inhibition of AR prevented LPS-induced activation of protein kinases upstream to NF-κB activation such as PKC and MAPK in HLECs. CONCLUSIONS. The results indicate that AR mediates the bacterial endotoxin signaling that could damage HLECs by regulating the signals that activate the redox-sensitive transcription factor NF-κB and cause inflammation. Thus, inhibition of AR could be a therapeutic target for Gram-negative bacterial infection-induced visual complications.

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